The Jun N-terminal kinase inhibitor SP600125 is a ligand and antagonist of the aryl hydrocarbon receptor.

Exposure of the immortalized human breast epithelial cell line MCF10A to the Jun N-terminal kinase (JNK) inhibitor anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) suppressed, in a concentration-dependent manner (IC50 is approximately 2 microM), the induction of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cotreatment with SP600125 also suppressed the accumulation of TCDD-induced nuclear aryl hydrocarbon receptor (AhR)-DNA complexes, as assessed by electrophoretic mobility shift assays. Concentrations of SP600125 < or = 50 microM did not transform the AhR into a DNA-binding species when added to rat liver cytosol. However, addition of SP600125 to cytosol just before TCDD addition completely suppressed AhR transformation and DNA binding (IC50 approximately 7 microM). Sucrose gradient analyses using rat liver and murine hepatoma 1c1c7 extracts demonstrated that SP600125 competed with TCDD for binding to the AhR. These results suggest that SP600125 is an AhR ligand and functions as an AhR antagonist at concentrations used to pharmacologically inhibit JNK.     

JNK inhibitor SP600125 reduces COX-2 expression by attenuating mRNA in activated murine J774 macrophages

Inducible prostaglandin synthase (cyclooxygenase-2, COX-2) is highly expressed in inflammation. The signaling mechanisms involved in the up-regulation of COX-2 are not known in detail. In the present study we investigated the role of c-Jun NH2-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family in COX-2 expression and prostaglandin (PG) E2 production in murine J774 macrophages activated by bacterial lipopolysaccharide (LPS). LPS caused a transient activation of JNK which was followed by increased COX-2 expression. Anthra(1,9-cd)pyrazol-6(2H)-one (SP600125), an inhibitor of JNK, inhibited phosphorylation of c-Jun with an IC50 of 5-10 microM. At the same concentrations SP600125 suppressed also LPS-induced COX-2 protein levels and PGE2 production. SP600125 did not alter LPS-induced COX-2 mRNA levels when measured 3 h after addition of LPS, whereas mRNA levels were significantly reduced in SP600125-treated cells when measured 24 h after addition of LPS. LPS-induced COX-2 mRNA levels reduced faster in cells treated with SP600125 than in control cells. Cycloheximide (that is known to activate JNK) enhanced COX-2 expression and its effect was inhibited by SP600125. The present results suggest that JNK pathway is involved in the up-regulation of COX-2 expression possibly by a mechanism related to the stability of COX-2 mRNA.     

c-Abl kinase regulates curcumin-induced cell death through activation of c-Jun N-terminal kinase

Curcumin, a natural phenolic compound found in turmeric (Curcuma longa) exhibits anticancer properties, attributed to its antiproliferative and apoptosis-inducing activity. The ubiquitously expressed nonreceptor tyrosine kinase c-Abl regulates stress responses induced by oxidative agents such as ionizing radiation and H2O2. In this study, we show that c-Abl is an important component of the cell death response activated by curcumin and that Abl mediates this response partly through activation of c-Jun N-terminal kinase (JNK). Therefore, inhibition of Abl by STI571 [imatinib (Gleevec)] treatment or down-regulation of Abl expression through Abl-specific short-hairpin RNA (shRNA) diminished cell death induction and JNK activation. Highlighting the interdependent nature of the Abl and JNK signaling in the curcumin-induced cell death response, a JNK inhibitor [anthra(1,9-cd)pyrazol-6(2H)-one-1,9-pyrazoloanthrone (SP600125)] caused very little cell death inhibition in STI571-pretreated cells and in Abl shRNA-expressing cells. Moreover, treatment with Abl and JNK inhibitor alone or together caused similar levels of cell death inhibition. Although p53 induction in response to curcumin treatment is dependent on Abl, we found that Abl-->p53 signaling is not necessary for curcumin-induced cell death. Taken together, the results demonstrate the differential roles played by Abl-->p53 and Abl-->JNK signaling events in modulating the cell death response to curcumin.     

Pro- and anti-apoptotic roles of c-Jun N-terminal kinase (JNK) in ethanol and acetaldehyde exposed rat hepatocytes

We have examined the significance of the activation of c-Jun N-terminal kinase (JNK) and p42/44 mitogen-activated protein kinase (MAPK) by ethanol and acetaldehyde in rat hepatocyte apoptosis. Acetaldehyde induced rapid and transient (15 min) activation of p42/44 MAPK followed by activation of JNK, which remained above control up to 1 h. Ethanol activated JNK for up to 4 h. Both ethanol and acetaldehyde caused apoptosis as determined by DNA fragmentation, caspase-3 activation and 2'[4-ethoxyphenyl]-5-[4-methyl-piperazinyl]-2,5'-bi-1H-benzimidazole (Hoechst 33342) staining. Ethanol-induced apoptosis was blocked by JNK inhibitor 1,9-pyrazoloanthrone (SP600125), indicating that JNK activation is pro-apoptotic. In contrast, acetaldehyde-induced apoptosis was not suppressed by this inhibitor. In fact, SP600125 potentiated acetaldehyde-induced apoptosis, suggesting that JNK activation is anti-apoptotic. Inhibition of p42/44 MAPK by MAPK kinase (MKK1) inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), potentiated apoptosis by acetaldehyde or ethanol, suggesting anti-apoptotic role of p42/44 MAPK. The activation of JNK by ethanol or acetaldehyde was insensitive to the genistein (tyrosine kinase inhibitor), GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide, protein kinase C [PKC] inhibitor) and N-acetylcysteine (N-AC) (antioxidant), whereas p42/44 MAPK activation by acetaldehyde was inhibited by genistein and GF109203X. Furthermore, p42/44 MAPK activation is not necessary for the JNK activation. In summary, transient activation of JNK by acetaldehyde is anti-apoptotic, whereas sustained activation of JNK by ethanol is pro-apoptotic. The activation of p42/44 MAPK appears to be anti-apoptotic for both ethanol and acetaldehyde. Thus, JNK activation by ethanol and acetaldehyde can be both pro- and anti-apoptotic in hepatocytes.     

Inflammatory pathway analysis using a high content screening platform

High content cellular screening assays are useful tools to investigate the interplay between signaling pathways and offer valuable platforms to determine the mode of action, potency, and selectivity of potential drug candidates in a biological setting. We describe a cell-based multiplex fluorescent imaging assay that permits concurrent detection and quantification of the distribution of nuclear factor kappaB (NFkappaB) p65/RelA and phosphorylated forms of p38 and c-Jun between the cytosol and nucleus. Cellular screening, data acquisition, and data interpretation were conducted on the ArrayScan HCS Reader (Cellomics Inc., Pittsburgh, PA). A significant window between untreated and interleukin-1alpha (IL-1alpha) stimulated HeLa cells for all three targets was achieved with low variability. Staining specificity was confirmed with blocking peptides and pharmacological inhibition of p38, c-Jun-N-terminal kinase (JNK), and inhibitory kappaB kinase 2, and channel bleed-through was eliminated or counterbalanced by the use of fixed exposure times together with careful reporter channel selection. The JNK inhibitor SP600125 was used as a demonstration compound because in addition to inhibiting nuclear accumulation of phosphorylated c-Jun it reduced nuclear translocation of phosphorylated p38 and NFkappaB p65/RelA in a dose-dependent manner, indicating a lack of SP600125 selectivity. This was supported by RNA interference where co-transfection of small interfering RNA targeting both JNK1 and JNK2, to limit signaling redundancy, significantly inhibited IL-1alpha-stimulated translocation of phosphorylated c-Jun without altering phosphorylated p38 and NFkappaB p65/RelA redistribution. This image analysis application is a valuable and information-rich screening tool to investigate compound selectivity and/or cross-talk between key signaling pathways involved in the inflammatory response.     

Critical role of c-Jun N-terminal protein kinase activation in troglitazone-induced apoptosis of human HepG2 hepatoma cells

The peroxisome proliferator-activated receptor agonist troglitazone (TRO) was used for treatment of non-insulin-dependent diabetes until its removal from the market because of its severe hepatotoxicity. However, the mechanism for its hepatotoxicity is still poorly understood. In this study, we investigated whether TRO caused cell death by altering signaling pathways associated with cell damage and survival in human hepatoma cells. Our data reveal that TRO caused time- and concentration-dependent apoptosis of HepG2 and Chang liver human hepatoma cells, as evidenced by DNA fragmentation and staining with Hoechst 33342. In contrast, 50 or 100 microM rosiglitazone, a structural analog of TRO, did not cause apoptosis in these hepatoma cells. TRO activated both c-Jun N-terminal protein kinase (JNK) and p38 kinase about 5-fold between 0.5 and 8 h before they returned to control levels at 16 h in HepG2 cells. In contrast, TRO failed to activate the extracellular signal-regulated kinase. Furthermore, TRO increased the levels of proapoptotic proteins, Bad, Bax, release of cytochrome c, and cleavage of Bid in a time-dependent manner. The antiapoptotic Bcl-2 protein level decreased in hepatoma cells treated with TRO. Pretreatment of hepatoma cells with a selective JNK inhibitor, anthra[1,9-cd]pyrazol-6(2H)-one (SP600125), significantly reduced the rate of TRO-induced cell death, whereas 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), an inhibitor of p38 kinase, had little effect on apoptosis. Pretreatment with SP600125 also prevented JNK activation and c-Jun phosphorylation. In addition, rosiglitazone, which is not as toxic to hepatoma cells as TRO, did not stimulate JNK activity. Transfection of cDNA for the dominant-negative mutant JNK-KR (Lys-->Arg) or SEK1-KR (Lys-->Arg), an immediate upstream kinase of JNK, significantly reduced TRO-induced JNK activation and cell death rate. Furthermore, SP600125 pretreatment effectively prevented the TRO-mediated changes in Bad, Bax, Bid cleavage, and cytochrome c release. These data strongly suggest that hepatotoxic TRO causes apoptosis by activating the JNK-dependent cell death pathway accompanied by increased Bid cleavage and elevation of proapoptotic proteins.     

Involvement of c-jun N-terminal kinase in G2/M arrest and caspase-mediated apoptosis induced by cardiotoxin III (Naja naja atra) in K562 leukemia cells.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, may have a potentiality as a structural template for rational drug design in killing cancer cells. Treatment of K562 cells with 0.3 microM of CTX III resulted in G2/M phase cell cycle arrest that was associated with a marked decline in protein levels of G2/M regulatory proteins including cyclin A, cyclin B1, Cdk2 and Cdc25C. In contrast to no effect on the phosphorylation of ERK, p38 MAPK and Akt, an activation of JNK was noted when K562 cells were exposed to CTX III. CTX III-mediated G2/M phase arrest and apoptosis were reduced by treatment with the JNK-specific inhibitor SP600125, but not by ERK and p38MAPK inhibitors. Further investigation showed that the specific JNK inhibitor, SP600125, reduced the activation of caspase-3, caspase-9, and reversed the decline in the expression of cyclin B1. Taken together, our data show for the first time that JNK, but not ERK, p38MAPK or Akt signaling, plays an important role in CTX III-mediated G2/M arrest and apoptosis in K562 cancer cells.     

JNK inhibitor SP600125 is a partial agonist of human aryl hydrocarbon receptor and induces CYP1A1 and CYP1A2 genes in primary human hepatocytes

SP600125, a specific inhibitor of c-Jun-N-Terminal kinase (JNK), was reported as a ligand and antagonist of aryl hydrocarbon receptor (AhR) [Joiakim A, Mathieu PA, Palermo C, Gasiewicz TA, Reiners Jr JJ. The Jun N terminal kinase inhibitor SP600125 is a ligand and antagonist of the aryl hydrocarbon receptor. Drug Metab Dispos 2003;31(11):1279-82]. Here we show that SP600125 is not an antagonist but a partial agonist of human AhR. SP600125 significantly induced CYP1A1 and CYP1A2 mRNAs in primary human hepatocytes and CYP1A1 mRNA in human hepatoma cells HepG2. This effect was abolished by resveratrol, an antagonist of AhR. Consistent with the recent report, SP600125 dose-dependently inhibited CYP1A1 and CYP1A2 genes induction by a prototype AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in human hepatocytes. Moreover, SP600125 displayed typical behavior of a partial agonist in HepG2 cells transiently transfected with a reporter plasmid containing two inverted repeats of the dioxin responsive element or with a plasmid containing 5'-flanking region of human CYP1A1 gene. SP600125 transactivated the reporter plasmids with EC(50) of 0.005 and 1.89 microM, respectively. On the other hand, TCDD-dependent transactivation of the reporter plasmids was inhibited by SP600125 with IC(50) values of 1.54 and 2.63 microM, respectively. We also tested, whether the effects of SP600125 are due to metabolism. Using liquid chromatography/mass spectrometry approach, we observed formation of two minor monohydroxylated metabolites of SP600125 in human hepatocytes, human liver microsomes but not in HepG2 cells. These data imply that biotransformation is not responsible for the effects of SP600125 on AhR signaling. In conclusion, we demonstrate that SP600125 is a partial agonist of human AhR, which induces CYP1A genes.     

The role of c-Jun N-terminal kinase in the A beta-mediated impairment of LTP and regulation of synaptic transmission in the hippocampus

The effects of the beta-amyloid peptide (Abeta) fragment 25-35 were investigated on hippocampal synaptic transmission and long-term potentiation (LTP) in vitro. Abeta([25-35]) was found to impair both post-tetanic potentiation (PTP) and LTP in the hippocampal CA1. The anthra[1,9-cd]pyrazol-6(2H)-one, SP600125, was used to inhibit c-Jun N-terminal kinase (JNK) activity, which is believed to mediate cell death. Prior application of SP600125 attenuated the Abeta([25-35])-mediated impairment of PTP and LTP, when measured from the pre-drug baseline. In the presence of SP600125 alone, we observed an increase in baseline synaptic transmission and reduction in paired-pulse facilitation, consistent with an increase in synaptic transmission. There was no alteration in the level of PTP and LTP obtained, when measured from the pre-drug baseline. In the presence of both SP600125 and Abeta, however, PTP was greatly enhanced compared with controls. We therefore suggest that the activation of the JNK signalling pathway mediates the effects of Abeta on synaptic plasticity. Our data also indicate that endogenous JNK activity may regulate neurotransmitter release in the hippocampal CA1 in vitro.     

C-jun N-terminal kinase modulates 1,25-dihydroxyvitamin D3-induced cytochrome P450 3A4 gene expression.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is known to induce the expression of cytochrome P450 3A4 (CYP3A4) in human colon carcinoma Caco-2 cells. Recently, it was demonstrated that the vitamin D receptor (VDR) regulates 1,25(OH)(2)D(3)-induced CYP3A4 gene expression through the xenobiotic-responsive element and the vitamin D-responsive element located on the 5'-flanking region of the CYP3A4 gene. On the other hand, we previously reported that protein kinases such as protein kinase C and tyrosine kinases contribute to the induction of CYP3A4 mRNA by 1,25(OH)(2)D(3). In the present study, we examined the involvement of mitogen-activated protein kinases (MAPKs) in the 1,25(OH)(2)D(3)-induced CYP3A4 gene expression using MAPK inhibitors. Curcumin, a c-Jun N-terminal kinase (JNK) pathway inhibitor, and anthra[1,9-cd]pyrazole-6(2H)-one (SP600125), a JNK inhibitor, suppressed the induction of CYP3A4 mRNA by 1,25(OH)(2)D(3), but not 2'-amino-3'-methoxyflavone (PD098059), a mitogen-activated protein kinase kinase-extracellular signal-regulated kinase (ERK) pathway inhibitor, or 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a p38 inhibitor. In addition, we demonstrated that SP600125 dose-dependently inhibited the CYP3A4 promoter activity induced by 1,25(OH)(2)D(3) using the reporter plasmid of the CYP3A4 promoter. However, SP600125 did not affect 1,25(OH)(2)D(3)-induced transactivation of the DR3 via VDR. These results indicate that JNK, but not ERK or p38, is required for the optimal activation of the CYP3A4 gene induced by 1,25(OH)(2)D(3).     

Low concentrations of paraquat induces early activation of extracellular signal-regulated kinase 1/2, protein kinase B, and c-Jun N-terminal kinase 1/2 pathways: role of c-Jun N-terminal kinase in paraquat-induced cell death.

Paraquat is a herbicide with a potential risk to induce parkinsonism due to its demonstrated neurotoxicity and its strong structural similarity to 1-methyl-4-phenylpyridinium (MPP(+)), a well-known neurotoxin which causes a clinical syndrome similar to Parkinson's disease (PD). However, at present very little is known about the signaling pathways activated by paraquat in any cell system. In this study, we have investigated the effect of paraquat on extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and protein kinase B (PKB) activation in E18 cells. Low concentrations of paraquat stimulated very early increases in ERK1/2, JNK1/2, and PKB phosphorylation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited early paraquat-induced increases in PKB phosphorylation. Furthermore, early paraquat-mediated increases in ERK1/2 activation were sensitive to the mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK1/2 responses were blocked by the JNK1/2 inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Pretreatment with wortmannin, LY 294002, or PD 98059 had no effect on paraquat cell death in E18 cells. In contrast, SP 600125 significantly decreased paraquat-induced cell death in E18 cells. In conclusion, we have shown that low concentrations of paraquat stimulate robust very early increases in ERK1/2, JNK1/2, and PKB phosphorylation in E18 cells. Furthermore, the data presented clearly suggest that inhibition of the JNK1/2 pathway protects E18 cells from paraquat-induced cell death and support the fact that inhibition of early activation of JNK1/2 can constitute a potential strategy in PD treatment.     

c-Jun N-terminal kinase contributes to norepinephrine-induced contraction through phosphorylation of caldesmon in rat aortic smooth muscle

Vascular smooth muscle contraction is mediated by activation of extracellular signal-regulated kinase (ERK) 1/2, an isoform of mitogen-activated protein kinase (MAPK). However, the role of stress-activated protein kinase/c-Jun N-terminal kinase (JNK) in vascular smooth muscle contraction has not been defined. We investigated the role of JNK in the contractile response to norepinephrine (NE) in rat aortic smooth muscle. NE evoked contraction in a dose-dependent manner, and this effect was inhibited by the JNK inhibitor SP600125. NE increased the phosphorylation of JNK, which was greater in aortic smooth muscle from hypertensive rats than from normotensive rats. NE-induced JNK phosphorylation was significantly inhibited by SP600125 and the conventional-type PKC (cPKC) inhibitor G?6976, but not by the Rho kinase inhibitor Y27632 or the phosphatidylinositol 3-kinase inhibitor LY294002. Thymeleatoxin, a selective activator of cPKC, increased JNK phosphorylation, which was inhibited by G?6976. SP600125 attenuated the phosphorylation of caldesmon, an actin-binding protein whose phosphorylation is increased by NE. These results show that JNK contributes to NE-mediated contraction through phosphorylation of caldesmon in rat aortic smooth muscle, and that this effect is regulated by the PKC pathway, especially cPKC.     

Potential role of c-Jun NH2-terminal kinase in allergic airway inflammation and remodelling: effects of SP600125

Asthma is a chronic inflammatory disease of the airways associated with structural changes such as increased airway smooth muscle mass, which may contribute to impairment of lung function. To determine whether c-Jun NH2-terminal kinase (JNK) of the mitogen-activated protein kinase signalling pathway participated in these changes, the effects of an inhibitor, SP600125 (anthra [1, 9-cd] pyrazole-6 (2H)-one), were examined in a murine model of chronic airway inflammation and remodelling. Mice sensitised to ovalbumin were exposed to ovalbumin aerosol and were treated with SP600125 [30 mg kg(-1) intraperitoneal (i.p.)] on days of exposure. SP600125 significantly reduced eosinophil and lymphocyte numbers in bronchoalveolar lavage fluid, suppressed eosinophilic inflammation within the bronchial submucosa, inhibited goblet cell hyperplasia, and increased airway smooth muscle cell number in allergen-exposed mice. SP600125 also inhibited allergen-induced increase in bronchial responsiveness. SP600125 inhibited JNK activity in the challenged lungs. Although SP 600125 may also have other effects, we conclude that c-Jun NH2-terminal kinase may play a role in allergen-induced inflammation and remodelling associated with bronchial hyperresponsiveness.     

Dexmedetomidine‐induced contraction involves c‐Jun NH2‐terminal kinase phosphorylation through activation of the 5‐lipoxygenase pathway in the isolated endothelium‐denuded rat aorta

Vasoconstriction induced by dexmedetomidine, a highly selective alpha‐2 adrenoceptor agonist, mainly involves c‐Jun NH₂‐terminal kinase (JNK) phosphorylation in the isolated endothelium‐denuded aorta. We carried out an in vitro study to determine the main arachidonic acid metabolic pathway that is involved in dexmedetomidine‐induced JNK activation. Cumulative dexmedetomidine concentration‐contractile response curves were generated in the endothelium‐denuded rat aorta in the presence or absence of the following inhibitors: the JNK inhibitor SP600125, the phospholipase A₂ inhibitor quinacrine dihydrochloride, the non‐specific lipoxygenase (LOX) inhibitor nordihydroguaiaretic acid, the 5‐LOX inhibitor AA‐861, the dual 5‐LOX and cyclooxygenase (COX) inhibitor phenidone, the non‐specific COX inhibitor indomethacin, the cytochrome p450 epoxygenase inhibitor fluconazole, the COX‐1 inhibitor SC‐560, and the COX‐2 inhibitor NS‐398. The effect of the alpha‐2 adrenoceptor inhibitor rauwolscine and other inhibitors, such as quinacrine dihydrochloride, nordihydroguaiaretic acid, AA‐861, phenidone, indomethacin and the protein kinase C inhibitor GF 109203X, on dexmedetomidine‐induced JNK phosphorylation was investigated in rat aortic vascular smooth muscle cells with western blotting. The effect of dexmedetomidine on 5‐LOX and COX‐2 expression was investigated in vascular smooth muscle cells. SP600125, quinacrine dihydrochloride, nordihydroguaiaretic acid, AA‐861, phenidone, rauwolscine and chelerythrine attenuated dexmedetomidine‐induced contraction. Indomethacin slightly attenuated dexmedetomidine‐induced contraction. Fluconazole and SC‐560 had no effect on dexmedetomidine‐induced contraction, whereas NS‐398 attenuated contraction. SP600125, rauwolscine, quinacrine dihydrochloride, nordihydroguaiaretic acid, AA‐861, phenidone and GF 109203X attenuated dexmedetomidine‐induced JNK phosphorylation. 5‐LOX and COX‐2 were upregulated by dexmedetomidine. Thus, dexmedetomidine‐induced alpha‐2 adrenoceptor‐mediated contraction is mediated mainly by 5‐LOX and partially by COX‐2, which leads to JNK phosphorylation.     

Inhibition of c-Jun N-terminal kinase sensitizes tumor cells to flavonoid-induced apoptosis through down-regulation of JunD

Reduction of susceptibility to apoptosis signals is a crucial step in carcinogenesis. Therefore, sensitization of tumor cells to apoptosis is a promising therapeutic strategy. c-Jun NH2-terminal kinase (JNK) has been implicated in stress-induced apoptosis. However, many studies also emphasize the role of JNK on cell survival, although its mechanisms are not completely understood. Previously, we found that inhibition of JNK activity promotes flavonoid-mediated apoptosis of human osteosarcoma cells. We thus determined whether inhibition of JNK sensitizes tumor cells to a bioflavonoid-induced apoptosis, and whether this effect of JNK is a general effect. As the results, quercetin and genistein as well as a flavonoid fraction induced apoptosis of tumor cells, which was further accelerated by specific JNK inhibitor, SP600125 or by small interfering RNA specific to JNK1/2. This effect was specific to types of cells because it was further apparent in tumorigenic cell lines. Inhibition of JNK by SP600125 also reduced flavonoid-stimulated nuclear induction of JunD which was known to have protective role in apoptosis, whereas JNK inhibition alone had little effect on apoptosis. The flavonoid-induced apoptosis of tumor cells was significantly enhanced by transfecting them with antisense JunD oligonucleotides. These results suggest that inhibition of JNK facilitates flavonoid-induced apoptosis through down-regulation of JunD, which is further sensitive to tumor cells. Therefore, combination with a specific JNK inhibitor further enhances the anti-cancer and chemopreventive potential of bio-flavonoids.     

Roles of p38 and JNK mitogen-activated protein kinase pathways during cantharidin-induced apoptosis in U937 cells

Cantharidin is an active compound from blister beetles traditionally used for the treatment of cancer. It is known to exert its antitumor activity by inducing apoptosis in cancer cells. However, its signaling pathway still remains unclear. Therefore, we investigated the roles of the mitogen-activated protein kinases (MAPKs) and the tumor suppressor gene, p53, during cantharidin-induced apoptosis in U937 human leukemic cells. Cantharidin effectively activated ERK-1/2, p38 and JNK in U937 cells in a time- and dose-dependent manner. Cantharidin also exhibited a strong cytotoxicity and induced apoptosis in U937 cells. For the evaluation of the role of MAPKs, PD98059, SB202190 and SP600125 were used as MAPK inhibitors for ERK-1/2, p38 and JNK. PD98059 did not affect cantharidin-induced cytotoxicity and apoptosis, whereas SB202190 and SP600125 significantly interfered with cytotoxic and apoptotic activities induced by cantharidin. Cantharidin alone induced the apoptosis by phosphorylation of p53, up-regulation of downstream target genes, MDM2 and p21 and also cleaved caspase-3, whereas SB202190 and SP600125 caused the down-regulation of p53, MDM-2, p21 and cleaved caspase-3 after a co-treatment with cantharidin. Similarly, SB202190 and SP600125 significantly disturbed the caspase-3 activity after a co-treatment with cantharidin by colorimetric assay. Taken together, these results suggest that cantharidin can induce apoptosis by activation of p38 and JNK MAP kinase pathways associated with p53 and caspase-3.     

Involvement of oxidative stress-activated JNK signaling in the methamphetamine-induced cell death of human SH-SY5Y cells

Methamphetamine (METH) is one of the most commonly abused drugs that may result in neurotoxic damage. Many lines of evidence have revealed that oxidative stress plays an important role in METH-induced neurotoxic effects. In a previous study, it was demonstrated in human neuroblastoma SH-SY5Y cells that enhanced oxidative stress was related to METH-induced apoptosis. To evaluate which of the three major mitogen-activated protein (MAP) kinase signaling pathways are involved in the process, namely the extracellular signal-related kinases (ERK), the p38 MAP kinases (p38) and the Jun-N-terminal kinases (JNK), we performed a time-course assessment. This indicated that METH induced an increase in the phosphorylation of ERK and JNK, but not of p38. Moreover, a JNK-specific inhibitor, SP600125, partially but significantly rescued METH-induced cell death, while PD98059 (an ERK kinase inhibitor) and SB203580 (a p38 inhibitor) had no protective effect. We also found that vitamin E (Vit E) prevented METH-induced JNK phosporylation and SP600125 inhibited METH-induced c-Jun phosphorylation. Furthermore, METH-activated caspase-3 activity was significantly repressed by Vit E and in SP600125 treated cells. We suggest that the oxidative stress-activated JNK signaling pathway is involved in METH-induced cell death.